The αLβ2 integrin LFA-1 plays a key role in T-cell adhesion to the endothelial vasculature and migration into both secondary lymphoid organs and peripheral tissues via interactions with its target protein ICAM-1, but the pathways that regulate LFA-1-mediated T-cell polarity and migration are not fully understood. In this study we screened two RNAi libraries targeting G protein-coupled receptors (GPCR)/GPCR-associated proteins and kinases in a HuT-78 T-cell line model of LFA-1-stimulated T cell migration. Based on staining of the actin cytoskeleton, multiple parameters to measure cell morphology were used to assess the contribution of 1109 genes to LFA-1-mediated T cell polarity and migration. These RNAi screens identified a number of both novel and previously identified genes that either increased or decreased the polarity and migratory capacity of these cells. Following multiparametric analysis, hierarchical clustering and pathway analysis, three of these genes were characterised in further detail using primary human T-cells, revealing novel roles for the heterotrimeric G protein subunit Gβ1 and casein kinase 2 in LFA-1-mediated T-cell polarity and migration in-vitro. Our studies also highlighted a new role for ICAP-1, an adaptor protein previously described to be associated with β1 integrins, in β2 integrin LFA-1-directed migration in T-cells. Knockdown of ICAP-1 expression in primary T cells revealed a role in cell polarity, cell velocity and transmigration towards SDF-1 for this adaptor protein. This study therefore uncovers new roles for GPCR/GPCR-associated proteins and kinases in T-cell migration and provides potential novel targets for modulation of the T-cell immune response

The α Lβ 2 integrin LFA-1 plays a key role in T-cell adhesion to the endothelial vasculature and migration into both secondary lymphoid organs and peripheral tissues via interactions with its target protein ICAM-1. Although members of the chemokine family of G-protein coupled receptors and heterotrimeric G proteins are known to influence LFA-1 activation and ICAM binding, the role of these receptors and their downstream effectors has not been fully characterised. In addition, a number of kinases are activated in response to LFA-1/ICAM-1 signalling in T-cells but the extent to which the kinome influences T-cell migration is not fully understood. In this study we screened two RNAi libraries targeting G protein-coupled receptors (GPCR)/GPCR-associated proteins and kinases in a HuT-78 T-cell line model of LFA-1-stimulated T cell migration. Based on staining of the actin cytoskeleton, multiple parameters to measure cell morphology were used to assess the contribution of 1109 genes to LFA-1-mediated T cell polarity and migration. These RNAi screens identified a number of both novel and previously identified genes that either increased or decreased the polarity and migratory capacity of these cells. Following multiparametric analysis, hierarchical clustering and pathway analysis, three of these genes were characterised in further detail using primary human T-cells, revealing novel roles for the heterotrimeric G protein subunit Gβ1 and casein kinase 2 in LFA-1-mediated T-cell polarity and migration in-vitro. Our studies also highlighted a new role for ICAP-1, an adaptor protein previously described to be associated with β1 integrins, in β2 integrin LFA-1-directed migration in T-cells. Knockdown of ICAP-1 expression in primary T cells revealed a role in cell polarity, cell velocity and transmigration towards SDF-1 for this adaptor protein. This study therefore uncovers new roles for GPCR/GPCR-associated proteins and kinases in T-cell migration and provides potential novel targets for modulation of the T-cell immune response.